Modulation of intracellular cyclic AMP (cAMP) levels has been shown to impact on a number of cellular processes underlying changes in protein phosphorylation state, regulation of ion channel conductance, and gene expression. The concentration of intracellular cAMP is principally controlled at the level of its synthesis through the hormonal regulation of adenylyl cyclase, the enzyme catalyzing the conversion of ATP to cAMP. Adenylyl cyclase activity is regulated by hormones that couple through heterotrimeric G proteins that when activated, dissociate into alpha and beta-gamma dimers; both alpha and beta-gamma are capable of regulating the cyclase. Nine isoforms of adenylyl cyclase, encoded by separate genes, have been identified to date, and have been shown to be regulated by individual G protein subunits in an isoform-specific fashion. An additional property of adenylyl cyclases is their ability to integrate multiple simultaneous hormonal inputs. The importance of proper adenylyl cyclase regulation is underscored by the identification of mutations in the receptor and G protein components found in a number of human disease states such as hyperfunctioning thyroid adenomas, pseudohypoparathyroidism and McCune-Albright syndrome. In the first aim the principal investigator proposes to examine the structural basis for the type-specific regulation of adenylyl cyclases by G protein beta-gamma and inhibitory Gi-alpha subunits. In the second aim, the principal investigator will use genetic and biochemical approaches to identify and characterize activating mutant alleles of adenylyl cyclase. In the third aim, he will examine the behavior of adenylyl cyclase mutants in cell culture systems, and determine the consequences of these mutations on intracellular cAMP regulation. In the final aim, the principal investigator will examine the possible involvement of adenylyl cyclase mutations in pathophysiological states and examine the oncogenic potential of activating mutant adenylyl cyclase alleles. These proposed studies should provide a significant understanding of the mechanisms underlying the regulation of adenylyl cyclases and in general, G protein-coupled effector systems, and will provide the basis for elucidating possible defects in adenylyl cyclase structure or function as the basis for abnormal signal transduction in human disease states.